Toll-like receptors (TLRs) are key components of innate immunity; they serve as the first line of defense against invading pathogens such as bacteria and viruses. The signaling initiated by TLRs is a double- edged sword. On the one hand, it may lead to confining or eliminating the invading organisms; on the other hand, a prolonged and exaggerated response can cause tissue and organ damage. Moreover, TLR signaling triggered by exogenous or endogenous ligands contributes to the pathogenesis of many chronic inflammatory diseases. For example, TLR2 and TLR4 are involved in atherosclerosis, autoimmune colitis, SLE, diabetes and Alzheimer's disease. Therefore, blockade of excessive TLR signaling is a therapeutic approach being pursued for these diseases. However, currently there are no approved TLR antagonists for clinic use. A Chinese herb, Sparganium stoloniferum has long been used in Traditional Chinese Medicine (TCM) for the treatment of several inflammatory diseases. Although much work has been done with extracts from this herb, no in-depth molecular investigation of its components has been performed. Recently, in an effort to isolate and functionally characterize single compounds from Sparganium stoloniferum tubers, we identified a novel compound, Sparstolonin B (SsnB) that selectively blocks TLR2- and TLR4-mediated signaling. This R21 proposal is aimed at continuing this exciting developmental research. The central hypothesis is that SsnB can be developed as an anti-inflammatory agent by virtue of its selective inhibitory effects on TLR2 and TLR4 signaling. To test this hypothesis, we propose two specific aims. SA1. To evaluate the toxicity and the anti- inflammatory efficacy of SsnB in vivo. We will first evaluate the in vivo toxicity of SsnB and then test if SsnB can suppress the inflammatory responses in endotoxemia and sepsis mouse models. SA2. To further elucidate the molecular mechanism by which SsnB blocks TLR2 and TLR4 signaling. We will identify the acting sites of SsnB on TLR2 and TLR4 signaling pathways. These studies will provide mechanistic insights into the therapeutic effects of Sparganium stoloniferum tubers in inflammatory diseases, translating a centuries-old alternative therapy modality into modern pharmacology. The identification and confirmation of SsnB as a TLR2 and TLR4 antagonist will provide an opportunity to develop a new anti-inflammatory agent. In future studies, we will test the therapeutic value of SsnB for several immune-related chronic inflammatory diseases such as atherosclerosis and diabetes. PUBLIC HEALTH RELEVANCE: There is an unmet need for the treatment of chronic inflammatory diseases. We have identified a Chinese herb-derived small molecule compound, SsnB, with selective TLR2 and TLR4 blocking activities. Elucidation of the molecular mechanism and confirmation of the in vivo anti-inflammatory activity will lead to the development of SsnB into a selective TLR2 and TLR4 antagonist and an anti-inflammatory agent.